JPS63216476A - Serum-free medium for freeze stocking - Google Patents
Serum-free medium for freeze stockingInfo
- Publication number
- JPS63216476A JPS63216476A JP62050950A JP5095087A JPS63216476A JP S63216476 A JPS63216476 A JP S63216476A JP 62050950 A JP62050950 A JP 62050950A JP 5095087 A JP5095087 A JP 5095087A JP S63216476 A JPS63216476 A JP S63216476A
- Authority
- JP
- Japan
- Prior art keywords
- medium
- serum
- cells
- free
- cryopreservation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000012679 serum free medium Substances 0.000 title abstract description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 52
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229920000609 methyl cellulose Polymers 0.000 claims abstract description 18
- 239000001923 methylcellulose Substances 0.000 claims abstract description 18
- 239000002577 cryoprotective agent Substances 0.000 claims abstract description 5
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 claims abstract description 4
- 235000010981 methylcellulose Nutrition 0.000 claims abstract 5
- 239000012595 freezing medium Substances 0.000 claims description 27
- 235000011187 glycerol Nutrition 0.000 claims description 7
- 239000002609 medium Substances 0.000 abstract description 36
- 210000004102 animal cell Anatomy 0.000 abstract description 9
- 210000004027 cell Anatomy 0.000 description 46
- 238000005138 cryopreservation Methods 0.000 description 9
- 239000007758 minimum essential medium Substances 0.000 description 9
- 238000007710 freezing Methods 0.000 description 8
- 230000008014 freezing Effects 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000010257 thawing Methods 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 210000003501 vero cell Anatomy 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- MKXKFYHWDHIYRV-UHFFFAOYSA-N flutamide Chemical compound CC(C)C(=O)NC1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 MKXKFYHWDHIYRV-UHFFFAOYSA-N 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、培養動物細胞を凍結状態で長期間保存する際
に必要な凍結保存用培地、特に血清を含まない培地に関
する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a cryopreservation medium necessary for storing cultured animal cells in a frozen state for a long period of time, particularly a serum-free medium.
培養動物細胞は、一般に継代培養中に諸種の性質が変化
しやすい。その変化は、細胞増殖能、物質生産能、染色
体数等にあられれる。このような細胞の性質の変化をで
きる限り回避し、安定した性質を示す細胞を繰り返し、
再現性よく使用するために、凍結状態で細胞を長期間保
存する技術は、学術研究、商業目的を問わず不可欠であ
る。このために使用される細胞凍結保存用の培地として
は、現在凍結障害防護剤としてグリセリン軸１ｙｃｅｒ
ｉｎ）またはジメチルスルホキシド（ｄｉｍｅｔｈｙｌ
ｓｕｌｆｏｘｉｄｅ 。Cultured animal cells generally tend to change various properties during subculture. The changes occur in cell proliferation ability, substance production ability, chromosome number, etc. We avoid such changes in cell properties as much as possible, and repeat cells that exhibit stable properties.
For reproducible use, techniques for preserving cells in a frozen state for long periods of time are essential for both academic research and commercial purposes. The cell cryopreservation medium used for this purpose currently includes glycerin-based 1ycer as a cryoprotective agent.
in) or dimethyl sulfoxide (dimethyl
sulfoxide.
以下ＤＭＳＯという）、並びに血清を含む培地が広く使
用されている。Media containing DMSO (hereinafter referred to as DMSO) and serum are widely used.
しかしながら、培養動物細胞の中には、血清を全く含ま
ない培地で培養されているものがあり、この無血清培養
細胞の凍結保存のためには、血清を含まない凍結保存用
培地の開発が望まれている。However, some cultured animal cells are cultured in a medium that does not contain serum at all, and in order to cryopreserve these serum-free cultured cells, it is desirable to develop a cryopreservation medium that does not contain serum. It is rare.
したがって本発明の目的は、無血清培養動物細胞の凍結
保存に適した、血清を含まない凍結保存用培地を提供す
ることである。Therefore, an object of the present invention is to provide a serum-free cryopreservation medium suitable for cryopreservation of serum-free cultured animal cells.
上記目的は、凍結障害防護剤として、メチルセルロース
と、ジメチルスルホキシドまたはグリセリンとを含む無
血清凍結保存用培地により達成される。The above object is achieved by a serum-free cryopreservation medium containing methylcellulose and dimethyl sulfoxide or glycerin as cryoprotectants.
本発明の無血清凍結保存用培地は下記の成分を含んでい
る。The serum-free cryopreservation medium of the present invention contains the following components.
（イ）培養動物細胞の種類に応じて使用される培地から
血清を除いたもの。これは一般に合成培地と称される。(b) Culture medium used depending on the type of cultured animal cells, with serum removed. This is commonly referred to as a synthetic medium.
例えば、イーグルの最少必須培地 （Ｅａｇｌｅｌｓ
ｍｉｎｉｍｕｍ ｅｓｓｅｎｔｉａｌ ｍｅｄｉｕｍ）
（以下ＭＥＭ培地という）、ハムのＦ−１２培地（Ｈａ
ｍ−ｓ Ｆ−１２ａ＋ｅｄｉｕｍ）、ＲＰＭ１１６４０
培地、ＤＭ−１６０培地、三橋の培地等である。For example, Eagle's Minimum Essential Medium (Eagles
minimum essential medium)
(hereinafter referred to as MEM medium), Ham's F-12 medium (Ha
m-s F-12a+edium), RPM11640
medium, DM-160 medium, Mitsuhashi medium, etc.
（ロ）メチルセルロース
（ハ）ＤＭＳＯまたはグリセリン
メチルセルロースの含有量は、好ましくは０．０２〜１
％（ｗ／ｖ）、さらに好ましくは０．０５〜０．２％（
ｗ／ｖ）である。０．０２％より少ないときは、メチル
セルロースによる凍結障害防護効果が不十分となり、１
％を越えると、凍結保存培地の粘性が高くなりすぎて、
実用的見地から好ましくない。(b) Methylcellulose (c) The content of DMSO or glycerin methylcellulose is preferably 0.02 to 1
% (w/v), more preferably 0.05 to 0.2% (
w/v). When it is less than 0.02%, the freezing damage protection effect of methylcellulose is insufficient, and 1.
%, the viscosity of the cryopreservation medium becomes too high;
Unfavorable from a practical standpoint.
ＤＭＳＯまたはグリセリンの含有量は、好ましくは１〜
２０％（ｖ／ｖ） 、さらに好ましくは５〜１５％（ｖ
／ｖ）である。１％以下では、基本的な凍結障害防護効
果があられれず、２０％を越えると、直接的な細胞障害
作用を示すようになり好ましくない。The content of DMSO or glycerin is preferably 1 to 1.
20% (v/v), more preferably 5-15% (v/v)
/v). If it is less than 1%, the basic effect of protecting against freezing damage will not be achieved, and if it exceeds 20%, it will show a direct cytotoxic effect, which is not preferable.
本発明の無血清凍結保存用培地は、たとえば以下のよう
にして使用される。The serum-free cryopreservation medium of the present invention is used, for example, as follows.
（ｉ）培養細胞を無血清凍結保存用培地に懸濁する。ま
たは培養基質に付着した状態の細胞が接する培地を無血
清凍結保存用培地に交換する。(i) Suspend cultured cells in serum-free cryopreservation medium. Alternatively, the medium in contact with the cells attached to the culture substrate is replaced with a serum-free cryopreservation medium.
（ｉｉ） −７０℃以下で凍結保存する。(ii) Store frozen at -70°C or below.
本発明の無血清凍結保存用培地は、すべての培養動物細
胞を長期間凍結保存することができるが、このような動
物細胞の例としては、Ｌ−Ｐ３細胞、ハイブリドーマ１
９３、ＨｅＬａ −Ｐ３ （ヒト子宮癌由来）、Ｌ５１
７８Ｙ（マウスリンパ腫細胞）などがあげられる。The serum-free cryopreservation medium of the present invention can cryopreserve all cultured animal cells for a long period of time, but examples of such animal cells include L-P3 cells and hybridoma 1.
93, HeLa-P3 (derived from human uterine cancer), L51
Examples include 78Y (mouse lymphoma cell).
以下実施例により本発明をさらに具体的に説明する。The present invention will be explained in more detail with reference to Examples below.
実施例Ｌ Ｌ−Ｐ３細胞の凍結保存（１）２ｇのメ
チルセルロースを１００ｍ１の純水に懸濁し、オートク
レーブで滅菌後、４℃で一夜撹拌、溶解する。Example L Cryopreservation of L-P3 cells (1) 2 g of methylcellulose is suspended in 100 ml of pure water, sterilized in an autoclave, and then stirred and dissolved overnight at 4°C.
（２）通常使用されるＤＭ−１６０培地の溶質を２倍濃
度に調製したＤＭ−１６０培地１容と（１）のメチルセ
ルロース液１容を混ぜ、さらにこの混合液に通常のＤＭ
−１６０培地６容とＤＭＳＯ２容を加える。これを２倍
濃度無血清凍結保存用培地とする。(2) Mix 1 volume of DM-160 medium prepared with twice the concentration of solutes in the commonly used DM-160 medium and 1 volume of the methylcellulose solution from (1), and add regular DM to this mixture.
- Add 6 volumes of 160 medium and 2 volumes of DMSO. This is used as a double concentration serum-free cryopreservation medium.
（３） 培養されているＬ−Ｐ３細胞の培地を除き、
スクレーパーにより細胞をはがす。(3) Remove the culture medium of L-P3 cells,
Peel off the cells with a scraper.
（４）軽くビベッテイグを行い、細胞を分散させる。(4) Bivet lightly to disperse the cells.
（５）細胞を１１００Ｏｒｐ、３分間の遠心分離によっ
て収集する。(5) Harvest cells by centrifugation at 1100 Orp for 3 minutes.
（６）収集した細胞を通常のＤＭ−１６０培地で１回洗
う。(6) Wash the collected cells once with regular DM-160 medium.
（７） この細胞を通常のＤＭ＝１６０培地に懸濁し、
細胞密度を２Ｘ１０’個／ｍβ前後に合わせる。(7) Suspend the cells in normal DM=160 medium,
Adjust the cell density to around 2×10′ cells/mβ.
（８） （２）で調製した２倍濃度無血清凍結保存用
培地を同量、または１／３量添加し、よく混合する。(8) Add the same amount or 1/3 amount of the double concentration serum-free cryopreservation medium prepared in (2) and mix well.
（９）凍結用アンプルに１ｍｉづつ分注し、プログラム
フリーザーに入れ、−１℃／ ｍ ｉ ｎの速度で一７
０℃以下にまで冷却し、凍結する。(9) Dispense 1 mm each into freezing ampoules, place in a program freezer, and freeze at a rate of -1°C/min.
Cool to below 0°C and freeze.
αＯ超低温フリーザーまたは凍結保存用液体窒素タンク
に保存する。Store in an αO ultra-low temperature freezer or liquid nitrogen tank for cryopreservation.
この方法によって２４時間凍結保存されたし・Ｐ３細胞
の生存率は表１に示されている。この細胞は、通常の無
血清培養で対数増殖期にあっても、若干の死細胞を細胞
集団中に含んでいる性質があるが、その細胞集団中の凍
結前の生存率はメチルセルロースによる影ｔを受けない
。メチルセルロースを含まない凍結保存用培地（ｃｏｎ
ｔｒｏｌ） に比べ、上記の無血清凍結保存用培地を
用いた場合は、凍結融解後も１．７倍以上の生存率を保
持している。The survival rate of P3 cells cryopreserved for 24 hours by this method is shown in Table 1. These cells have the property of containing some dead cells in the cell population even in the logarithmic growth phase in normal serum-free culture, but the viability of the cell population before freezing is affected by methylcellulose. I don't receive it. Methylcellulose-free cryopreservation medium (con
trol), when the above serum-free cryopreservation medium is used, the survival rate is 1.7 times higher even after freezing and thawing.
表Ｌ Ｌ−Ｐ３細胞の凍結保存
最終細胞密度２Ｘ１０’／ｍｊｉ！で凍結し、２４時間
後解凍した。解凍後、細胞を通常の培地で一回洗浄し、
０．０６％のトリパンブルーを含む通常の培地を加え、
２分間放置した。その後、リン酸緩衝生理食塩水で一度
洗浄し、血球計算盤で総細胞数と、トリパンブルーで染
色されなかちた生細胞を計数し、生存率を産出した。Table L L-P3 cell cryopreservation final cell density 2X10'/mji! It was frozen and thawed 24 hours later. After thawing, cells were washed once with regular medium;
Add normal medium containing 0.06% trypan blue,
It was left for 2 minutes. Thereafter, the cells were washed once with phosphate buffered saline, and the total number of cells and viable cells that were not stained with trypan blue were counted using a hemocytometer to calculate the survival rate.
対照は１０％のＤＭＳＯのみを含むＤＭ−１６０培地、
Ｎα１は０．１％メチルセルロースと１０％ＤＭＳ○を
含むＤＭ−１６０培地、Ｎα２は０．０５％メチルセル
ロースと５％Ｄ Ｍ Ｓ Ｏヲ含（ｊ Ｄ Ｍ−１６０
培地である。Control was DM-160 medium containing only 10% DMSO;
Nα1 is a DM-160 medium containing 0.1% methylcellulose and 10% DMS○, Nα2 is a medium containing 0.05% methylcellulose and 5% DMS○ (j DM-160
It is a medium.
実施例２ ハイブリドーマ１９３−（１）の凍結保存
（１）実施例１の（１） 、（２）に従って２倍濃度無
血清凍結保存用培地を作製する。但しＤ Ｍ −１６０
培地の代わりにＭＥＭ培地を使用する。Example 2 Cryopreservation of Hybridoma 193-(1) (1) A double concentration serum-free cryopreservation medium is prepared according to (1) and (2) of Example 1. However, DM-160
Use MEM medium instead of culture medium.
（２） この２倍濃度無血清凍結保存用培地１容と、
無血清培地ＳＦＭＩ ０１で培養されているハイブリド
ーマ１９３−（１）細胞浮遊液１容を混合する。(2) 1 volume of this double concentration serum-free cryopreservation medium,
Mix 1 volume of hybridoma 193-(1) cell suspension cultured in serum-free medium SFMI 01.
（３）凍結用アンプルに１ｍｌづつ分注し、プログラム
フリーザーに入れ、−１℃／ｃｏ ｉ ｎの速度で一７
０℃以下にまで冷却し、凍結する。(3) Dispense 1 ml into freezing ampoules, place in a program freezer, and freeze at a rate of -1°C/coin.
Cool to below 0°C and freeze.
αＱ 超低温フリーザーまたは凍結保存用液体窒素タン
クに保存する。αQ Store in an ultra-low temperature freezer or liquid nitrogen tank for cryopreservation.
この方法によって２４時間凍結保存された細胞の回収率
、生存率は表２に示されている。この細胞は凍結しなく
ても１０％ＤＭＳＯを含む培地に懸濁しただけで生存率
が低下する傾向を示す性質があるが、凍結融解後は、メ
チルセルロースを含まない凍結保存用培地（対照）に比
べ、上記の無血清凍結保存用培地を用いた場合は、凍結
融解後も１０％血清添加凍結保存用培地とほぼ同等の細
胞回収率、生存率を保持している。The recovery rate and survival rate of cells cryopreserved for 24 hours by this method are shown in Table 2. Even if these cells are not frozen, their survival rate tends to decrease just by suspending them in a medium containing 10% DMSO. In comparison, when the above-mentioned serum-free cryopreservation medium is used, the cell recovery rate and survival rate are maintained almost the same as the cryopreservation medium supplemented with 10% serum even after freezing and thawing.
表２ ハイブリドーマ１９３−（１）の凍結保存最終
細胞密度１０”／＋ｎｊ！で凍結し、２４時間後解凍し
た。細胞数はコールタ−カウンターで計数した。コール
タ−カウンターの設定値は、粒子直径９．４２５μｍ
（下限）〜１９．３６μｍ （上限）である。細胞の回
収率は、解凍時、破壊されずに原形をとどめている細胞
を計数しくこれを総細胞数とした）、メチルセルロース
を含まない無血清凍結保存用培地（１０％のＤＭＳＯを
含むＭＥＭ培地、対照）を１００％としたときの相対値
であられしである。このなかから死細胞を除去するため
別に一部の細胞懸濁液をとり、＃濃度８００ｕｎｉｔ／
＋ｎｊ！のデスパーゼ（東洋醸造）を添加し３７℃１時
間インキュベートした。死細胞はこの処理により上記の
設定値にセットされたコールタ−カウンターでは計数さ
れなくなる。生存率は、こうして計数された生細胞数を
総細胞数で除し％で表したものである。Table 2 Cryopreservation of Hybridoma 193-(1) It was frozen at a final cell density of 10"/+nj! and thawed after 24 hours. The number of cells was counted with a Coulter counter. The setting value of the Coulter counter was a particle diameter of 9 .425μm
(lower limit) to 19.36 μm (upper limit). The cell recovery rate was determined by counting the cells that remained intact during thawing and using this as the total number of cells), serum-free cryopreservation medium that does not contain methylcellulose (MEM medium containing 10% DMSO) , control) is taken as 100%. Separately take a part of the cell suspension from this to remove dead cells, #concentration 800 units/
+nj! Despase (Toyo Jozo Co., Ltd.) was added and incubated at 37°C for 1 hour. This process prevents dead cells from being counted by the Coulter counter set to the above settings. The survival rate is the number of living cells thus counted divided by the total number of cells and expressed as a percentage.
対照はメチルセルロースを含まｆｌＯ％ＤＭｓｏヲ含む
ＭＥＭ培地、０．１％ＭＣは０．１％のメチルセルロー
スと１０％のＤＭＳＯを含むＭ Ｅ Ｍ培地、１０％Ｆ
ＢＳは１０％のウシ胎児血清と１０％のＤＭＳＯを含む
ＭＥＭ培地である。Control is MEM medium containing methylcellulose and flO% DMSO, 0.1% MC is MEM medium containing 0.1% methylcellulose and 10% DMSO, 10% F
BS is MEM medium containing 10% fetal bovine serum and 10% DMSO.
実施例３ 顕微鏡観察試験用Ｖｅｒｏ細胞の凍結保存（
１）実施例１の（１）、（２）に従って２倍濃度無血清
凍結保存用培地を作製する。但しＤＭ−１６０培地の代
わりにＭＥＭ培地を、また、ＤＭＳＯの代わりにグリセ
リンを使用する。この培地のｐＨを炭酸ガスにておよそ
７．０〜７．４に調整しておく。Example 3 Cryopreservation of Vero cells for microscopic observation test (
1) Prepare a double concentration serum-free cryopreservation medium according to (1) and (2) of Example 1. However, MEM medium is used instead of DM-160 medium, and glycerin is used instead of DMSO. The pH of this medium is adjusted to approximately 7.0 to 7.4 using carbon dioxide gas.
（２） この２倍濃度無血清凍結保存用培地１容と、
ｐＨを７．２に調整したＭ Ｅ Ｍ培地１容を混合する
。(2) 1 volume of this double concentration serum-free cryopreservation medium,
Mix 1 volume of MEM medium adjusted to pH 7.2.
（３） あらかじめ無血清のＭ Ｅ Ｍ培地で少なく
とも６時間培養されているカバーガラス上のＶｅｒｏ細
胞を、そのまま凍結用のプラスチック容器に移し、（２
）で作製した無血清凍結保存用培地１ｍｊ！を加え、こ
の培地が細胞面を覆うように静置する。(3) Vero cells on a cover glass, which have been cultured in serum-free MEM medium for at least 6 hours, are transferred as they are to a plastic container for freezing.
) serum-free cryopreservation medium 1mj! Add this medium and let stand so that the cell surface is covered.
（４） この姿勢のまま、プログラムフリーザーに入
れ、−１℃／ ｍ ｉ ｎの速度で一７０℃以下にまで
冷却し、凍結する。(4) Place in a program freezer in this position and cool to below -70°C at a rate of -1°C/min to freeze.
（５） 超低温フリーザーまたは凍結保存用液体窒素
タンクに保存する。(5) Store in an ultra-low temperature freezer or liquid nitrogen tank for cryopreservation.
この方法によって２４時間凍結保存された細胞の解凍後
の生存率は悪く、凍結前あった４８９６００個の細胞の
うち生着していた細胞は２．５％にすぎなかった。しか
し生着していた細胞は１００倍程度の倍率でも顕微鏡観
察下で簡単に視野内に捕えることができ、十分顕微鏡観
察試験用に使用しうるちのとなっている。Cells cryopreserved for 24 hours using this method had a poor survival rate after thawing, with only 2.5% of the 489,600 cells that had survived before being frozen. However, the engrafted cells can be easily captured within the field of view under microscope observation even at a magnification of about 100 times, making them suitable for use in microscopic observation tests.
これに対して、メチルセルロースを含まないほかは全く
同一の培地を用いて、同じ細胞を２４時間凍結保存した
場合には、生着していた細胞は１％未満であり、生着細
胞を１００倍程度の倍率の５微鏡観察下で視野内に捕え
ることは極めて困難であった。In contrast, when the same cells were cryopreserved for 24 hours using the same medium that did not contain methylcellulose, less than 1% of the cells were engrafted, which was 100 times the number of engrafted cells. It was extremely difficult to capture it within the field of view under 5-microscope observation at a magnification of about 100 yen.
本発明の無血清凍結保存用培地によれば、血清を全く含
まない培地で培養されている無血清培養動物細胞を、凍
結状態で長期間安定に保存することができる。According to the serum-free cryopreservation medium of the present invention, serum-free cultured animal cells that are cultured in a medium that does not contain any serum can be stably stored in a frozen state for a long period of time.
Claims (3)
メチルスルホキシドまたはグリセリンとを含むことを特
徴とする無血清凍結保存用培地。(1) A serum-free cryopreservation medium characterized by containing methylcellulose and dimethyl sulfoxide or glycerin as a cryoprotectant.
％（ｗ／ｖ）である特許請求の範囲第１項記載の無血清
凍結保存用培地。(2) Methyl cellulose content is 0.02 to 1.0
% (w/v) of the serum-free cryopreservation medium according to claim 1.
が、１〜２０％（ｖ／ｖ）である特許請求の範囲第１項
または第２項記載の無血清凍結保存用培地。(3) The serum-free cryopreservation medium according to claim 1 or 2, wherein the content of dimethyl sulfoxide or glycerin is 1 to 20% (v/v).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62050950A JPS63216476A (en) | 1987-03-05 | 1987-03-05 | Serum-free medium for freeze stocking |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62050950A JPS63216476A (en) | 1987-03-05 | 1987-03-05 | Serum-free medium for freeze stocking |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63216476A true JPS63216476A (en) | 1988-09-08 |
| JPH058675B2 JPH058675B2 (en) | 1993-02-02 |
Family
ID=12873102
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62050950A Granted JPS63216476A (en) | 1987-03-05 | 1987-03-05 | Serum-free medium for freeze stocking |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63216476A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0646840A (en) * | 1992-08-04 | 1994-02-22 | Nippon Zenyaku Kogyo Kk | Solution for freezing and storing cell |
| WO2002000848A1 (en) * | 2000-06-28 | 2002-01-03 | Ascendia Ab | Dna/rna-embedding medium and method of use |
| JP2004504003A (en) * | 1999-09-24 | 2004-02-12 | モルフォゲン ファーマスーティカルズ インコーポレイテッド | Pluripotent embryonic-like stem cells, compositions, methods and uses thereof |
| WO2008007463A1 (en) * | 2006-07-12 | 2008-01-17 | Nippon Zenyaku Kogyo Co., Ltd. | Composition for diluting and storing sperm |
| EP1950283A1 (en) * | 2005-11-17 | 2008-07-30 | Nippon Zenyaku Kogyo Co., Ltd. | Aqueous solution for cell preservation |
| JP2011507530A (en) * | 2007-12-21 | 2011-03-10 | ビン エム．アブドゥラー，ジャファー アリ | Protein-free gamete and embryo handling media products and culture media products |
-
1987
- 1987-03-05 JP JP62050950A patent/JPS63216476A/en active Granted
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0646840A (en) * | 1992-08-04 | 1994-02-22 | Nippon Zenyaku Kogyo Kk | Solution for freezing and storing cell |
| JP2004504003A (en) * | 1999-09-24 | 2004-02-12 | モルフォゲン ファーマスーティカルズ インコーポレイテッド | Pluripotent embryonic-like stem cells, compositions, methods and uses thereof |
| WO2002000848A1 (en) * | 2000-06-28 | 2002-01-03 | Ascendia Ab | Dna/rna-embedding medium and method of use |
| JP2004506414A (en) * | 2000-06-28 | 2004-03-04 | アセンデイア アクチエボラグ | DNA / RNA integration media and methods of use |
| US6706499B2 (en) | 2000-06-28 | 2004-03-16 | Ascendia Ab | DNA-embedding medium and method of use |
| EP1950283A1 (en) * | 2005-11-17 | 2008-07-30 | Nippon Zenyaku Kogyo Co., Ltd. | Aqueous solution for cell preservation |
| EP1950283A4 (en) * | 2005-11-17 | 2013-04-24 | Nippon Zenyaku Kogyo Co Ltd | Aqueous solution for cell preservation |
| US8460926B2 (en) | 2005-11-17 | 2013-06-11 | Nippon Zenyaku Kogyo Co., Ltd | Aqueous solution for cell preservation |
| KR101368924B1 (en) * | 2005-11-17 | 2014-03-04 | 니뽄젠야쿠코교 가부시키가이샤 | Aqueous solution for cell preservation |
| WO2008007463A1 (en) * | 2006-07-12 | 2008-01-17 | Nippon Zenyaku Kogyo Co., Ltd. | Composition for diluting and storing sperm |
| JP5238501B2 (en) * | 2006-07-12 | 2013-07-17 | 日本全薬工業株式会社 | Semen diluted storage composition |
| JP2011507530A (en) * | 2007-12-21 | 2011-03-10 | ビン エム．アブドゥラー，ジャファー アリ | Protein-free gamete and embryo handling media products and culture media products |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH058675B2 (en) | 1993-02-02 |
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